Obesity is a well-established risk factor for a number of diseases, including type 2 diabetes and coronary heart disease. Although weight loss is the most effective treatment for type 2 diabetes, current methods for reducing weight typically are insufficient for long-term weight loss. The mission of AdipoGenix, Inc. is to discover, develop and license novel therapeutics acting at the level of the fat cell (adipocyte) for the treatment of obesity and related disorders. Effective therapeutics, particularly those acting on the fat cell, is lacking. AdipoGenix already has developed advanced methods for culturing and differentiating human preadipocytes, and has established and validated a primary screening assay to monitor lipid accumulation in these cells. Using this assay 70,000 compounds were screened, and a new class of compounds that reduces lipid accumulation in differentiated human preadipocytes was identified. The present goal is to determine the mechanism of action by which these new compounds reduce lipid accumulation in order to facilitate further development of this class of compounds and to expand proprietary protection of our leads. In Aim 1 sufficient quantities of the lead and two related compounds will be synthesized for work in Aims 2 and 3. Radiolabeled lead compound also will be synthesized for use in Aim 2. In Aim 2 specific binding in homogenates from differentiated human preadipocytes using the unlabeled and radiolabeled compounds generated in Aim 1 will be determined. General receptor and mechanism of action screens also will be performed to identify the receptor and elucidate the cellular target mechanism by which these compounds reduce lipid accumulation in human adipocytes. In Aim 3 metabolic pathway analysis will be performed. Using this approach this new class of compounds has been shown not to act through PPARgamma or by stimulating lipolysis. Also in Aim 3, signaling pathways will be defined by analysis of focused signaling-pathway expression arrays using mRNA from differentiated human preadipocytes treated with our compounds. This work will produce key information to expedite receptor identification, determine mechanism of action, facilitate full lead optimization for the further development of this class of compounds, and to develop additional, new antiobesity drugs acting at the level of the fat cell.